Bladder cancer, or vesical cancer, is the second most frequent tumor of the genitourinary tract after prostate cancer [Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002; 52:23-47]. In a global context, it represents approximately 3 and 1%, in men and women, respectively, of all the deaths due to cancer. In absolute values, this means that about 95,000 men and about 35,000 women die every year due to this pathology. The ratio between incidence and death is different depending on the degree of development of each country. As extreme examples, it could be mentioned that in the North America area this ratio would be close to 0.2, whereas in sub-Saharan regions it would increase up to 0.6 [Edwards B K, Brown M L, Wingo P A et al. Annual report to the nation on the status of cancer, 1975-2002, featuring population-based trends in cancer treatment. J Natl Cancer Inst 2005; 97:1407-27; Pisani P, Parkin D M, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999; 83:18-29].
Unlike other tumors, familial genetic predisposition factors have virtually not been detected for the moment. In contrast, several environmental factors strongly related to bladder tumors have been detected. One of the most important factors, not only due to its relation to the disease but also due to its incidence in the population, is smoking. It has been observed that smokers have a risk three times higher than non-smokers of developing a bladder tumor. In fact, one third of bladder tumors are associated to tobacco consumption. Unfortunately, the carcinogenic agents present in tobacco have still not been clearly identified [Burch J D, Rohan T E, Howe G R et al. Risk of bladder cancer by source and type of tobacco exposure: a case-control study. Int J Cancer 1989; 44:622-28; Zeegers M P, Kellen E, Buntinx F, van den Brandt P A. The association between smoking, beverage consumption, diet and bladder cancer: a systematic literature review. World J Urol 2004; 21:392-401].
Different types of disorders can be found in the bladder at cell level. There are benign changes such as epithelial hyperplasias, urothelial metaplasias and Von Brunn's nests, among others. In contrast, dysplasias would correspond to disorders that are more or less intermediate between normal epithelium and carcinoma. Finally, different types of urothelial carcinomas are found in the bladder, which can divided into adenocarcinomas, squamous tumors and transitional cell carcinomas (TCC).
More than 90% of bladder tumors are TCCs. At the time of their diagnosis, approximately 75% are superficial tumors, 20% are invading muscular layers (infiltrating or invasive TCCs) and 5% are already metastatic. Of the superficial cases, approximately 20% are cured by means of a single surgical intervention, whereas between 50 and 70% recur one or more times after surgery, but never become infiltrating tumors. Between 10 and 30% of these superficial tumors become infiltrating tumors. These tumors are aggressive, poor-prognosis tumors with a mortality after 5 years of 50% and in the metastasized cases, the mortality after two years is 100% [Sanchez-Carbayo M, Socci N D, Charytonowicz E et al. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes. Cancer Res 2002; 62:6973-80; Adshead J M, Kessling A M, Ogden C W. Genetic initiation, progression and prognostic markers in transitional cell carcinoma of the bladder: a summary of the structural and transcriptional changes, and the role of developmental genes. Br J Urol 1998; 82:503-12; Babaian R J, Johnson O F, Llamas L, Ayala A G. Metastases from transitional cell carcinoma of urinary bladder. Urology 1980; 16:142-44].
The genetic pathways of superficial and invasive TCCs, although related, seem to be quite different. The most usual progression in superficial tumors seems to be hyperplasia, atypia and finally low-grade papillary TCCs. In invasive tumors, it is most usual to progress from an atypia to a dysplasia, to then pass to a tumor in situ (Tis) and end in an infiltrating tumor [Knowles M A. What we could do now: molecular pathology of bladder cancer. Mol Pathol 2001; 54:215-21].
Current diagnosis systems are based on a combination of urinary cytology (from squamous cells in urine) and of the direct observation of the bladder by means of cystoscopy. The latter is actually the main diagnostic and follow-up technique for tumors. It is performed by transurethral route, therefore it is an invasive and rather unpleasant technique for the patients. The sensitivity and specificity of this technique were believed to be quite high, although improvements in the actual technique (fluorescence cystoscopy) indicate that this is probably not so and that part of the recurrence observed in superficial tumors could be due to the lack of total resection in non-visible parts thereof [Jones J S. DNA-based molecular cytology for bladder cancer surveillance. Urology 2006; 67:35-45]. Urinary cytology is in turn a non-invasive diagnostic technique with a high sensitivity and specificity for high-grade tumors. However, this technique shows limitations for detecting low-grade tumors [Bastacky S, Ibrahim S, Wilczynski S P, Murphy W M. The accuracy of urinary cytology in daily practice. Cancer 1999; 87:118-28]. Furthermore, the interpretation of the cytology is highly observer-dependent, therefore they may be inter-observer differences, especially in low-grade tumors.
All these limitations have led to the search for more reliable non-invasive bladder cancer markers. Finding a non-invasive marker with a high sensitivity and specificity for bladder TCC would be very helpful for clinical practice. In fact, several studies describe new tumor markers in urine, such as the test for the bladder tumor antigen NMP22 [Wiener H G, Mian C, Haitel A, Pycha A, Schatzl G, Marberger M. Can urine bound diagnostic tests replace cystoscopy in the management of bladder cancer? J Urol 1998; 159:1876-80; Soloway M S, Briggman V, Carpinito G A et al. Use of a new tumor marker, urinary NMP22, in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J Urol 1996; 156:363-67], fibrin degradation products [Schmetter B S, Habicht K K, Lamm D L et al. A multicenter trial evaluation of the fibrin/fibrinogen degradation products test for detection and monitoring of bladder cancer. J Urol 1997; 158:801-5.], telomerase [Takihana Y, Tsuchida T, Fukasawa M, Araki I, Tanabe N, Takeda M. Real-time quantitative analysis for human telomerase reverse transcriptase mRNA and human telomerase RNA component mRNA expressions as markers for clinicopathologic parameters in urinary bladder cancer. Int J Urol 2006; 13:401-8], tests based on fluorescent in situ hybridization [Hailing K C, King W, Sokolova I A et al. A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine. J Urol 2002; 167:2001-6] or flow cytometry [Takahashi C, Miyagawa I, Kumano S, Oshimura M. Detection of telomerase activity in prostate cancer by needle biopsy. Eur Urol 1997; 32:494-98; Trott P A, Edwards L. Comparison of bladder washings and urine cytology in the diagnosis of bladder cancer. J Urol 1973; 110:664-66], but although most of them have a higher sensitivity than urinary cytology, the latter is still the most specific [Bassi P, De M, V, De Lisa A et al. Non-invasive diagnostic tests for bladder cancer: a review of the literature. Urol Int 2005; 75:193-200].
It is known that many and very varied genetic disorders are found in urothelial tumors, therefore the current tendency is to search for genetic markers (either at the DNA, RNA or protein level) which can indicate the presence of carcinomas in the analyzed sample. Furthermore, it would be very interesting to be able to discriminate the aggressiveness of the tumor of a patient with these same markers, as this would allow a much more personalized and effective treatment. Finally, some of these markers could be possible therapeutic targets for developing new drugs to combat cancer.
Until recently, the capacity to analyze gene expression patterns was limited to a few genes per experiment. New technologies, such as DNA microarrays have completely changed the scenario. Thousands of genes can currently be analyzed in a single assay [Duggan D J, Bittner M, Chen Y, Meltzer P, Trent J M. Expression profiling using cDNA microarrays. Nat Genet 1999; 21:10-14; Granjeaud S, Bertucci F, Jordan B R. Expression profiling: DNA arrays in many guises. Bioessays 1999; 21:781-90]. Therefore, massive expression results of all tumor types have started to appear in literature, including bladder tumors [Sanchez-Carbayo M, Socci N D, Charytonowicz E et al. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes. Cancer Res 2002; 62:6973-80; Ramaswamy S, Tamayo P, Rifkin R et al. Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sci USA 2001.98:15149-54; Sanchez-Carbayo M, Socci N D, Lozano J J et al. Gene discovery in bladder cancer progression using cDNA microarrays. Am J Pathol 2003; 163:505-16; Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C. Tumor suppressor role of KiSS-1 in bladder cancer loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003; 162:609-17; Dyrskjot L, Thykjaer T, Kruhoffer M et al. Identifying distinct classes of bladder carcinoma using microarrays. Nat Genet 2003; 33:90-96], although most of the results have not been made public in their entirety. However, up until now, the studies which have been conducted with specific bladder cancer markers have been focused on one or on very few genes [Olsburgh J, Hamden P, Weeks R et al. Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol 2003; 199:41-49; Fichera E, Liang S, Xu Z, Guo N, Mineo R, Fujita-Yamaguchi Y. A quantitative reverse transcription and polymerase chain reaction assay for human IGF-II allows direct comparison of IGF-II mRNA levels in cancerous breast, bladder, and prostate tissues. Growth Horm IGF Res 2000; 10:61-70; Simoneau M, Aboulkassim T O, LaRue H, Rousseau F, Fradet Y. Four tumor suppressor loci on chromosome 9q in bladder cancer: evidence for two novel candidate regions at 9q22.3 and 9q31. Oncogene 1999; 18:157-63].
Given that the nature of these tumors is very heterogeneous, it does not seem very likely to be able to identify all or most carcinomas with a single marker. Thus, to be able to characterize most tumors it seems to be essential to combine several of the best markers to some type of extent.
In addition, although the direct analysis of urothelial tissue is the most comfortable alternative for developing a routine diagnostic method, it would be very interesting, as has been mentioned above, that said method were not invasive, because the latter decrease the quality of life of the patients and represent a much higher economic burden for healthcare.
Bladder fluids (urine or bladder washing) which are in contact with the entire bladder epithelium, and therefore with the tumor mass, seem to be a good alternative for detecting tumor markers, given that they represent an easy and non-invasive way to obtain the sample to be analyzed. Thus, a large number of works have been focused on the study of tumor markers in urine in the search for a non-invasive diagnostic method for bladder TCC. In fact, different tests with this objective have been marketed (NMP22, UroVysion, ImmunoCyt, Accu-Dx, etc.).
One alternative, which has still not been marketed, is the detection of bladder TCC in urine samples by means of determining the gene expression of bladder cancer markers. In fact, there are some studies suggesting the usefulness of this methodology, although they have been conducted with one or a few marker genes [Parekattil S J, Fisher H A, Kogan B A. Neural network using combined urine nuclear matrix protein-22, monocyte chemoattractant protein-1 and urinary intercellular adhesion molecule-1 to detect bladder cancer. J Urol 2003; 169:917-20; Eissa S, Kenawy G, Swellam M, El Fadle A A, Abd El-Aal A A, El Ahmady O. Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma. Clin Biochem 2004; 37:803-10; Larsson P C, Beheshti B, Sampson H A, Jewett M A, Shipman R. Allelic deletion fingerprinting of urine cell sediments in bladder cancer. Mol Diagn 2001; 6:181-88].
In response to these needs, the inventors, after an important research work, have identified 14 bladder tumor marker genes, from which they have developed a bladder cancer diagnosis and prognosis method based on the detection and quantification of the gene expression of these genes by means of quantitative real-time PCR in RNA extracted from bladder fluids, and their subsequent computer combination by means of an “alarm system”.